UNIT

Solutions, Dilutions and Concentrations

Developed by Nathan Garcia and Lynne Lovelace

Lesson 3: Determining the optimal salinity for the fecundity (hatching success) of brine shrimp Artemia.

Objective: Students will gain a better understanding of simple concepts in algebra as well as chemistry, biology and statistics. This lesson will also teach students about the marine environment and some of the marine organisms that live in it.

Materials and Equipment:

Graduated cylinders 100ml, 50ml, 25ml, 10ml

Artificial sea salt

Salt Refractometer (see picture, preferred but not necessary $52 www.technika.com)

Dissecting microscope (monocular = $72 stereo = $149 www.hometrainingtools.com)

Scale (triple beam balance)

Micro-well plate (see picture)

Pipettor (1 ml)

1 ml pipette tips

Brine Shrimp eggs ($11 www.brineshrimpdirect.com)

100 ml distilled water

Background:

The common brine shrimp (Artemia) is in the phylum Arthropoda, class Crustacea. Artemia are zooplankton like Copepods and Daphnia, which are often used as live food in an aquarium. The Artemia life cycle begins by the hatching of dormant cysts or eggs, which contain metabolically inactive embryos. The cysts can remain dormant for many years as long as they are kept dry and oxygen free. When the cysts are placed back into salt water they are re-hydrated and resume their development. Artemia cysts are best stored in a tightly sealed container in a cool dry environment, if possible, vacuum packed. The refrigerator is usually best.

Brine shrimp are well known for their ability to survive in extremely salty water. This organism can reproduce by two modes. One way is by viviparous or live reproduction or by oviparous or egg-laying reproduction. Typically these shrimp will have live reproduction in low salinity levels. However, when the salinity level is high, they lay eggs instead. Brine eggs will have different hatch success at different salinities. In order to determine the hatching success we will have to make solutions of various salinities and add a specific number of eggs to each solution. The percent of eggs that hatch in each solution will determine the hatching success. The results of the experiment will determine the optimal salinity for the hatching success of the brine shrimp

The salinity of seawater is typically in the range of about 33-35 parts per thousand (ppt). One part per thousand means that there is one gram of salt per 1000 grams of water. One ml of water weighs one gram. This may seem extremely convenient but this occurrence is no coincidence. The definition of a milliliter and a gram are based on the physical property known as the density of water.

So 100 ml of water will weigh 100 grams. So if we want to make artificial seawater that is 35 part per thousand (ppt) we will have to add 3.5 grams of our artifical sea salt. If we had 1000 ml (1 L) of water it would weigh 1000 g (1 Kg) and we would have to add 35 grams of our artifical sea salt to get an artificial sea water solution of 35 ppt.

Have students prepare seawater with the following salinity values:

Group 1	Group 2	Group 3	Group 4
150 ppt	50 ppt	35 ppt	20 ppt
100 ppt	45 ppt	30 ppt	10 ppt
75 ppt	40 ppt	25 ppt	5 ppt

Note that all of the salinity regimes listed in the table are not required to get a good understanding. What is important is that the range of salinities is covered and students learn how to prepare solutions using a serial dilution method. Also eggs may hatch the best around 30-35 ppt which is the reason for so regimes selections around this range.

Give group one 15 g of salt, group 2 should receive 5 g, Group 3 gets 3.5 grams of salt, and Group 4 gets 2 grams of salt. Start with 100 ml of distilled water and add 14 grams of sea salt. Next ask students to determine what they should do in order to make a solution of 120 ppt. Once this is done, dispense 5 ml of each solution in to the micro-well plate and put 10 brine shrimp eggs in each well. Let the well sit until the next day and analyze.

Next ask students to make conclusions about their results. In what concentration did eggs tend to hatch the most? In other words, out of 10 eggs in each salinity what percentage hatched?

Applications

Students next should learn how to present the data they acquired in a way that is easy to understand. A graph may be the easiest but most importantly, students should be allowed to come up with their own creative way of representing the material in a scientific manner. A table is another way to present the data.

Personally, I prefer a graph and students may also find that a graph is fairly simple and easy to understand. What ever method students choose it should be imperative that students invent their own way of making a graph or a table, creativity is part of the fun of science.

Here’s an example.

You may get completely different results than this! This is just a guess at what really will be the case. Also you may want to combine results of different groups of students so that the average includes more data points. This would be an excellent way to teach students what the average actually is, how it’s calculated and why it’s important. Another statistical measure that students can grasp at this point is the mode which is the highest point in the data set. In this example the data set has two modes, one is at 40 ppt and the other is at 30 ppt. Lastly another statistical measure you can teach is the range. Say there were three groups of students that performed the same experiment and they wanted to compile their data. One of the groups had 60% hatching success at 75 ppt. Another group had 50% hatching success at 75 ppt and the third group had only 10% hatching success at 75 ppt. This would average to 40 % hatching success but the range would be 10% to 60% hatching success at 75 ppt. It may be fun to graph the ranges along with the averages to give students a clear understanding of what the data actually shows.

Next students should recognize that although hatching success may be low at high salinity levels, these organisms, nonetheless, are able to survive and cope with the salinity stress. Hopefully students will begin to wonder how they are able to cope and what it means to deal with this type of stress in other marine organisms as well.

Ask students if there are any other environmental factors that we could test that might effect the fecundity of brine shrimp eggs. TEMPERATURE and LIGHT may be important. If you really want to be creative in the lesson, try using the same table of salinity regimes at different temperature or light regimes.